Abstract (inglese)

A major impediment in Hepatitis C Virus (HCV) research and drug development has been the lack of a culture system supporting virus production. This obstacle was recently overcome by using JFH1-based full-length genomes that allow the production of infectious viruses both in vitro and in vivo. Despite such improvement, the system was restricted to two structural gene sequences (JFH1 and J6), both derived from the genotype 2a, limiting comparative studies between different HCV strains. The system was thus extended by the creation of a series of inter-genotypic HCV chimeras that allow the production of infectious genotypes 1a, 1b and 3a particles, even if with a less efficiency in terms of productivity compared with the wild type JFH1 strain.
In the present study, based on the JFH1 strain, we generated two novel chimeric HCV constructs in which only the sequences encoding for the E1 and/or E2 glycoprotein ectodomains are substituted from JFH1 to the Con1 strain (genotype 1b). The entire structural region is maintained of the infectious strain JFH1, with the exception of the soluble portion of E1 and/or E2, allowing comparative analysis of the impact of such regions on virus morphogenesis.
Both JFH1/Con1E1E2 and JFH/Con1E2 chimeric constructs are able to replicate in hepatic cells. Importantly, we demonstrate for the first time that the E1E2 heterodimer formation is not hampered by the ectodomain swapping since glycoproteins from genotypes 2a and 1b can correctly interact each other. Nevertheless, none of the chimeric constructs allow the production of infectious viral particles. This evidence first suggests a specific role of E1 and E2 glycoproteins in HCV particle morphogenesis, and it is corroborated by several experiments. In particular we examined the JFH/Con1E2 construct, in which only the E2 ectodomain region is swapped from genotype 2a to 1b, by confocal microscopy analysis, trans-complementation experiments and by evaluating the presence of HCV infectious particle precursor within transfected cells. Overall our data provide strong evidences that the E2 ectodomain is involved in the HCV assembly through a genotype-specific interplay with the remaining viral structural proteins. As consequence, in the case of inter-genotypic chimeras, genetic incompatibility between JFH1 backbone and the E2 structural protein of genotype 1b dramatically affects the production of viral particles in our system. One of the most likely involved protein in such interactions seems to be NS2, the role of which was recently demonstrated as crucial in the HCV assembly/release processes. However this remains a hypothesis that needs to be verified in order to define the relationships among HCV proteins.